Objectives
Connect DNA replication to structure of genetic material
Describe the role of individual protein in DNA replication
Know what the telomere is and why its special
Describe the role of several telomere-binding proteins in
telomere function
Describe the role of telomerase in cellular immortality
Articulate ways that telomere biology can be used for
diagnostics, therapies, and biotechnology
Importance of DNA Replication
• DNA must be replicated every time a cell divides
– Errors in replication can cause mutation, thus
replication must be accurate; fidelity
– Eukaroytic genomes are large, therefore replication
must be fast and efficient
• Replication takes advantage of the “instructive”
aspect of base-pairing in DNA synthesis
– incorporation of nucleotide only occurs if proper
Watson-Crick base pairs can be made
Replication is Semi-Conservative
Much is Conserved in DNA Replication
Direction
Template
Primer
DNA Replication Steps
Initiation:
Assembly of a replication fork (bubble)
Fork is generated primosome
Elongation:
Addition of bases
Catalyzed by the replisome.
Parental strands unwind and daughter strands are synthesized
Termination:
Duplicated chromosomes are separated
The Replication Fork
Initiation: Work of the Primosome
At origins of replication the primosome goes to work
Helicase
Separates the DNA Strands
Single-strand DNA binding proteins
Bind strands in this region as single-stranded
Primase
Makes the primers
Helicase
Unwinds DNA to present template
Unwinding at the fork supercoils
DNA in advance of the fort
Topoisomerase relaxes supercoil
(ATP dependent)
Initiation: Isolate Template; Make Primer
ssDNA-Binding proteins keep template single stranded
Primase makes an RNA primer
Elongation: The Work of DNA Polymerase
Chemical Mechanism of Nucleotide Addition
Elongation: Rolling Circle
Two polymerase moving in opposite directions
Leading and Lagging Synthesis are Coupled
http://www.youtube.com/watch?v=5VefaI0LrgE&feature=related
Semi-Discontinuous Synthesis and Primer Removal
Priming leading strand
Leading strand synthesis
Priming lagging strand
Primers removed
Okazaki fragment synthesis
Resulting DNA fragments joined
Okazaki fragment synthesis
How Can Polymerase Copy The Whole Chromosome in a Few Hours?
Normal DNA Replication Fails to
Replicate the Telomere
How to Overcome the End-Replication Problem
Nosek, J.; Kosa, P.; Tomaska, L.BioEssays (2006) 28:182–190
The Telomere is the Chromosome Aglet
FISH analysis of
healthy chromosomes
FISH analysis of
chromosomes with
short telomeres
FISH analysis of
damaged chromosomes
Telomeres can
be visualized
Telomeres signal
is decreased
Chromosomes are fused
and damaged
The Chromosome End
3’ Overhang
...TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3’
...AATCCCAATCCCAATC-5’
Telomeric DNA, TTAGGG in humans
Double stranded region is 5,000 to 20,000 bases
Single strand region is 150-300 bases
Human Chromosome
The Chromosome Ends in a Loop
5’
5’
5’
3’
3’
3’
Griffith, J.D. et al. Cell 1999, 97, 503-14.
The Chromosome Ends in a Loop
5’
5’
5’
3’
Electron micrograph of a telomere
3’
3’
Griffith, J.D. et al. Cell 1999
The Telomere is a Protein-DNA Complex
5’
3’
POT1
5’
3’
TIN2
TRF1
5’
TPP1
TRF2
RAP1
3’
de Lange Genes Dev. 2005
The Telomere is a Protein-DNA Complex
5’
5’
5’
3’
5’
5’
5’
3’
POT1
TPP1
TIN2
TRF1
3’
TRF2
RAP1
3’
3’
3’
The Telomere is a Protein-DNA Complex
5’
5’
5’
3’
3’
3’
Nikitina and Woodcock J. Cell. Biol. 2004
5’
5’
5’
3’
3’
3’
Normal DNA Replication Fails to
Replicate the Telomere
Telomerase is a
Ribonucleoprotein Complex
Ciliate telomerase
Human telomerase
TERT
hTERT
Template
Dyskerin
p43 (p65)
´
Template
Common features of all telomerase complexes:
RNA supplies the template
TERT is the catalytic subunit
RNA-binding protein is present
The Telomere is the Chromosome Aglet
FISH analysis of
healthy chromosomes
FISH analysis of
chromosomes with
short telomeres
FISH analysis of
damaged chromosomes
Telomeres can
be visualized
Telomeres signal
is decreased
Chromosomes are fused
and damaged
Telomere Length Determines
Cellular Longevity
Time/cell divisions
Why Have Linear Chromosomes Anyway?
Meioses with a Circular Chromosome.
Ishikawa, F; Naito T. Mutation Research 434 1999. 99–107
Why Have Linear Chromosomes Anyway?
Telomere Maintenance and Human Health
Normal aging, stress, and the environment can all reduce telomere length
Stem cells senescence
wound healing rate decreases
tissue regeneration ability decreases
Aging disorders: deficiencies in telomere maintenance
Mutations in hTR, hTERT and DKC1 gene
Haplodefeciency in telomerase activity
Telomere Maintenance and Human Health
Cancer and telomere maintenance
Cancer cells require proper telomere maintenance
Telomerase positive (85%), ALT (15%)
Telomere length and telomerase correlations to prognosis
Stem cell technology
Attempts to maintain cells in culture without senescence
Cancer Requirements
Normal cell
Continuous growth
ras
Hyperproliferative cell
Overcome replicative senescence
Rb, p53
Proliferative cell
On verge of genetic crisis
Maintain telomeres
telomerase, ALT
Cancer
Telomere Length Determines
Cellular Longevity
Telomerase: Functions Away
From the Telomere
Inhibits apoptosis
Increases tumorigenicity
Promotes proliferation
Promotes healing
Promotes hair growth (in mice)
Therapeutic Approaches
Direct telomerase inhibition as anti-cancer modality
Delayed effect
In situ generation of a dominant negative complex
Unknown effects
Combination therapy: Telomerase and another target
Maybe useful for preventing remission and metastasis
Immunotherapy using anti-TERT antibodies: phase II clinical
Anticancer therapy by changing telomere sequence:
Instantaneous effect, but is it plausible?
Diagnostics and prognosis testing: TERT expression related
to cancer progression
GRN163L Enters the Clinic
5’-Palm-TAGGGTTAGACAA-NH2
Palm = Palmitoyl
NPS
GRN163L Inhibits Telomerase,
Telomere Synthesis, and Cell Growth
GRN163L Inhibits Tumor Growth
Enzyme Inhibitors
COOH
H
N
O
BIBR1532, from Boehringer Ingelheim
N
O
NH2
N
PPO
N3
N
N
Lavelle et al. Crit Rev Onc/Hem 2000, 34, 111-126
Inhibition of Telomerase Causes Telomere
Shortening, which can be Reversed
Shortening upon treatment
COOH
H
N
O
BIBR1532, from Boehringer Ingelheim
Damm et al. EMBO J 2001, 20, 6958-6968; Pascolo et al. J. Biol. Chem. 2002,
Inhibition of Telomerase Causes Telomere
Shortening, which can be Reversed
Shortening upon treatment
Halting treatment: regain telomeres
COOH
H
N
O
BIBR1532, from Boehringer Ingelheim
Damm et al. EMBO J 2001, 20, 6958-6968; Pascolo et al. J. Biol. Chem. 2002,
Combination Therapy